JP2002528519A - Pharmaceutical composition containing DNA encoding an antigenic protein having antitumor effect - Google Patents

Abstract

  (57) [Summary] Provided herein are suitable excipients for inducing an anti-tumor Ag-specific immune response, including one or more DNA molecules encoding fragments of a protein that is overexpressed in tumor cells. And a pharmaceutical composition combined with an adjuvant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a pool of DNA plasmid constructs containing the sequence of the fragment encoding human MUC-1 and to a sequence encoding a protein consisting of human ubiquitin, which fragment itself is fused to a bacterial LacI fragment. It concerns a pool of DNA plasmids preceded. The invention further relates to their use in preparing pharmaceutical compositions for use as anti-tumor DNA vaccines.

[0002]

[Prior art]

The present invention provides anti-tumor treatments based on the induction or activation of an immune response that can lead to tumor rejection. The validity of such an idea is demonstrated by the results of initial clinical trials; for example, patients treated with a viral vaccine containing a sequence encoding the cancer embryo antigen (CEA) will have an immune system activity against this antigen (Ts
ang KY et al. J. Natl. Cancer. Inst. 87: 982, 1995).

[0003] Activation of an immune anti-tumor response can be achieved in four different approaches: a) ex vivo processing of patient tumor cells to make them more immunogenic and preferred as vaccines; b) in vitro immune response Ex vivo processing of the patient's immune cells to pre-activate the tumor-associated antigens c) naked or encapsulated in liposomes or incorporated into viral particles (retrovirus, adenovirus, etc.). Coded DN
Inoculation of A; d) can be achieved through treatment with a recombinant or synthetic soluble tumor antigen conjugated or mixed with an adjuvant.

The first two approaches, which consist of processing the cells of every single patient, are limited in that they are absolutely patient-specific, while the latter two are comparable to conventional medicaments. It is intended to obtain the product.

The new vaccination method reflects the development of new technologies. Suggestions from experiments with DNA-exposed vaccines that induce a sustained antibody or cellular immune response are:
Traditional protein subunit vaccines consisting of certain specific peptides that induce lymphocyte populations are obsolete. Intramuscularly or intradermally injected proteins, encoded by exposed DNA, induce cytotoxic-specific responses as well as helper responses. This powerful combination is very effective, but the mechanisms involved are not yet fully understood. Muscle cells express only low levels of class I MHC antigens and do not appear to express class II antigens or costimulatory molecules. Thus, transfected muscle cells cannot play an important role in initiating the immune response itself. Recent data indicate that antigen presenting cells (APCs), such as macrophages or dendritic cells, are fundamental in the capture of monocyte-released antigen and subsequent processing and presentation of peptides involved in the background of class I and class II molecules. It plays a role, that is, inducing the activation of CD + cells with cytotoxic activity and the activation of CD4 + cells in cooperation with B lymphocytes in inducing an antibody response (Corr M et al J. EX
P. Med. 184: 1555, 1996) (Tighe, H. et al. Immunology Today 19:89, 1998).
.

[0006] Furthermore, the use of cytokines is known to improve the therapeutic effects derived from immunization with DNA. As reported in Irvine et al., J. Immunol. 156: 238, 1996, cytokines can be processed in the formation of foreign proteins. One alternative approach is represented by co-inoculation of a plasmid encoding the tumor antigen or the desired cytokine, ie, by producing the cytokine in situ (Kim JJ et al. Immunol 158: 816, 1997).
.

The active immunization approach of the present invention is based on the use of DNA vectors as vaccines against MUC-1 human antigen or polymorphic epithelial mucin (PEM), which is overexpressed in tumor cells. MUC-1 is an epithelial luminal surface glycoprotein (Patton S. et al. BBA 1241: 407, 1995). In the cell transformation process, this glycoprotein loses tip localization and its expression level increases dramatically. The function of the protein consists, for example, in the protection of the luminal surface in mammalian glands, ovaries, endometrium, colon, stomach, pancreas, bladder, kidney and the like. A disadvantage of glycosylation has been reported to be that tumor cell-associated MUC-1 is antigenically distinct from normal cell-associated MUC-1. This phenomenon exposes tumor MUC-1 to antigen epitopes that are normally masked by the sugar moiety in MUC-1 expressed in normal cells. This feature makes tumor MUC-1 particularly interesting in inducing a tumor-specific antibody response (Apostolopoulos V. et al. Crit. Rev. Immu
nol. 14: 293, 1994).

[0008] In contradiction, vaccination aims to induce an immune response against tumor cells expressing high levels of MUC-1, while protecting epithelial tissues with low expression levels. DNA vaccination relies on the entry of a gene or its part in the body's cells, followed by the transcription and translation of the inserted sequence, and consequently the intracellular synthesis of the corresponding protein. An important advantage of this system is that newly synthesized proteins are naturally processed intracellularly and the resulting peptides are linked to major histocompatibility complex class I molecules (MHC-I). Therefore, the MHC / peptide complex is, of course, exposed to the cell surface recognized by the immune system CD8 + cytotoxic cells. Only polypeptides synthesized in the cell are then processed and presented in association with MHC class I molecules,
Therefore, it makes it the only mechanism that stimulates a specific cytotoxic response. Vaccination systems based on protein or peptide treatment are mostly more effective at stimulating antibody immune responses, which to date have been shown to be ineffective in rejecting tumor cells. Current gene therapy techniques rely on DNA packaging into recombinant viral vectors (retroviruses and adenoviruses). Exposed DNA treatment is more advantageous in that it is more effective and safer than viral vector treatment (Kumar V and Sercarz E. N.
ature Med. 2: 857, 1966; McDonnel WM et al., New England J. of Med. 334:
42, 1996). In fact, exposed DNA is neither replicated nor integrated into host tissue DNA, and does not elicit an immune response to viral proteins.

The use of ubiquitin to enhance the processing of newly synthesized proteins and thus the induction of cytotoxic lymphocytes was recently reported (Rodriguez F. et al.,
J. Virology 71: 8497, 1997). N which makes them unstable and promotes decomposition
The use of nobiquitin to make proteins with terminal amino acids has been previously reported (Bechmair A. et al., SCIENCE 234: 179, 1986). The high degree of instability of these proteins is subsequently involved in the intracellular processing and presentation of model proteins by MHC-1 (Grant EP et al., J. Immunol. 155: 3750, 1).
995) (Wu Y and Kipps TJ, J. Imunol. 159: 6037, 1997).

[0010] In DNA vaccination, a single construct (influenza virus nucleoprotein) comprising a DNA fragment partially encoding an antigen, which has a higher plateau presentation effect compared to the whole analog of the antigenic sequence Has been reported (Anton
LC et al., J. Immunol. 158: 2535, 1997). Furthermore, the processing of intracellular proteins and the presentation of the respective peptides by MHC class I proteins under physiological conditions underlies the mechanism of immune surveillance. A specific protein and M
In the context of HC, there are peptide fragments called dominant (ie, dominant over subdominants and potential), and they generate any immune response because they are recognized as "self." Absent. In accordance with aspects of the present invention, it has emerged that approaches aimed at supporting non-dominant epitope presentation by processing a mixture of antigenic protein fragments can elicit a surprising cytotoxic immune response. Have been.

[0011]

Description of the Invention A DNA molecule encoding a fragment of a protein that is overexpressed in tumor cells can be conveniently used to induce an antigen-specific anti-tumor immune response I now know that.

[0012] The invention particularly relates to pharmaceutical compositions comprising one or more DNAs encoding a mucin (MUC-1) protein fragment.

The DNA used in the present invention may be a plasmid or a viral DNA, preferably a plasmid D obtained using the pMRS30 expression vector described in FIG.
NA.

[0014] The composition of the invention preferably comprises at least two DNA fragments of mucin (MUC-1) or another protein that is overexpressed in tumor cells.

The compositions of the present invention are preferably 200 to 700 nucleotides each, each sequence aligned, and about 50 to about 150 nucleotides are adjacent 3 ′ and / or 5 nucleotides. At the 'end, contains at least four fragments that may partially overlap.

[0016] The DNA fragment of the present invention may be preceded by DNA encoding ubiquitin at the 5 'end, and may be preceded by the LacI portion of Escherichia coli.

The present invention also relates to novel DNA fragments and the use of the above defined mucin-1 fragments in the preparation of medicaments and antitumor vaccines.

[0018]

[Means for Solving the Problems]

DETAILED DESCRIPTION OF THE INVENTION In eukaryotic cells, D encoding a fragment of the MUC-1 human protein antigen
An NA plasmid pool was prepared. The construct is described in FIG.
O95 / 11982, EM with accession number J05581
Based on the mammalian expression vector designated pMRS30, which contains a partial sequence of the MUC-1 cDNA reported in the BL database. DNA encoding MUC-1 was fragmented such that each fragment represented a separate part and partially overlapped with an adjacent one. Administration of such a mixture of plasmids can result in different plasmids for transfecting APC cells at different sites of administration. Thus, such cells produce and process distinct portions of the MUC-1 protein that provide the relevant peptides. In such conditions, the resulting subdominant and potential peptides can be presented in association with class I MHC molecules, ie, create a cytotoxic immune response.

The present invention relates to a group of four constructs comprising MUC-1 subsequences (FIGS. 1 to 4) in a mixture comprising at least two MUC-1 cDNA subfragments, and separately or to a MUC-1 cDNA subunit. The protein sequence containing ubiquitin and the LacI part of Escherichia coli used in the mixture containing the fragments (FIG. 6)
)), The use of a group of four constructs (FIGS. 7-10) containing the MUC-1 cDNA partial fragment preceded by the DNA encoding

The present invention relates to a construct comprising the nearly complete sequence of the MUC-1 cDNA (FIG. 5) and a nearly complete MUC-1 cDNA preceded by a DNA encoding a protein sequence comprising the ubiquitin and the LacI portion of Escherichia coli. It also relates to the use of the construct containing the complete sequence (FIG. 11).

Four constructs containing a partial sequence of the MUC-1 cDNA, and four constructs containing a partial sequence of the MUC-1 cDNA preceded by DNA encoding a protein sequence containing the ubiquitin and the LacI portion of Escherichia coli A mixture of the above mixtures represents a preferred embodiment of the present invention.

The constructs of the invention can be used in anti-tumor treatment of patients affected by a tumor characterized by high MUC-1 expression.

The construct described in the present invention was obtained as follows.

For the first series of constructs, MUC-1 DNA was obtained from the BT20 cell line by RT-PCR or by DNA partial chemical synthesis. Such a fragment was cloned into the pMRS30 expression vector and confirmed by sequencing.

In the case of the second series of constructs, fragments were obtained from the first series of constructs by PCR reamplification. These fragments are ubiquitin (mRN7 of MCF7 cell line).
A) and a partial LacI sequence (obtained from the commercial vector p
(Obtained by PCR from GEX). The DNA sequence thus obtained was then cloned into the pMRS30 expression vector and confirmed by sequencing.
For the intended therapeutic or prophylactic use, for example, The Immunologist, 1994, 2: 1; W
O 90/11092, Proc. Natl. Acad. Sci. USA, 1986, 83, 9551; US 5580859; I
mmunology today 19 (1998), 89-97; Proc. Natl. Acad. Sci. USA, 90 (199
3), 11478-11482; Nat.Med. 3 (1997), 526-532; Vaccine 12 (1994), 1495-14
98; DNA Cell. Biol. 12 (1993), 777-783.
Fragments or constructs of the invention are preferably formed using the methods previously employed for NA vaccines. Dosages will be determined based on clinical and pharmacological-toxicological tests. Generally, it may contain from 0.005 μg / kg to 5 μg / kg of the fragment mixture.
The composition of the present invention may also include a cytokine or a plasmid encoding the cytokine.

[0026]

EXAMPLES The present invention is further described by the following examples. Example 1 Construction of Plasmid pMRS166 BT20 tumor cells (ATCC HTB-19) were cultured in minimal essential Eagle's medium supplemented with 10% fetal calf serum. Trypsinize 10 million cells,
After washing with PBS, mRNA was extracted.

An aliquot of this RNA is synthesized with the following synthetic oligonucleotides:

[0028]

Embedded image

Was subjected to an RT-PCR (reverse transcriptase-polymerase chain reaction) reaction in the presence of

The resulting DNA fragment, which has been purified and digested with the restriction enzyme XbaI, is
The clone was cloned into the pMRS-30 expression vector containing the human cytomegalovirus promoter and the β-globin polyadenylation signal as claimed in 9511982. The resulting pMRS166 vector contains the ATG codon, EM
Includes a sequence corresponding to nucleotides 136-139 of BL sequence J05581 and a DNA fragment containing the two stop codons TGA and TAA. This fragment is shown in FIG.

Example 2 Construction of Plasmid pMRS169 An aliquot of the RNA obtained as reported in Example 1 was prepared using the following synthetic oligonucleotides:

[0032]

Embedded image

Amplified by RT-PCR in the presence of

The resulting DNA fragment purified and digested with restriction enzymes SmaI and XbaI was
A completely synthetically constructed DNA fragment, which partially corresponds to nucleotides 457-720 of the EMBL sequence J05581 and two stop codons TGA
And a DNA fragment containing TAA at the SmaI site. That is, the entire fragment was cloned into the XbaI site of the pMRS30 expression vector. The resulting pMR
The S169 vector contains DNA containing the ATG codon, two stop codons TGA and TAA, which partially correspond to nucleotides 205-720 of the EMBL sequence J05581. This fragment is shown in FIG.

Example 3 Construction of Plasmid pMRS168 An aliquot of the RNA obtained as reported in Example 1 was prepared using the following synthetic oligonucleotides:

[0036]

Embedded image

Amplified by RT-PCR in the presence of

The resulting DNA fragment, which had been purified and digested with the restriction enzyme XbaI, was digested with pMRS-3.
0 expression vector. The resulting pMRS168 vector contains a DNA fragment containing the ATG codon, a sequence corresponding to nucleotides 631-1275 of the EMBL sequence J05581, and two stop codons, TGA and TAA. This fragment is shown in FIG.

Example 4 Construction of Plasmid pMRS167 An aliquot of the RNA obtained as reported in Example 1 was prepared using the following synthetic oligonucleotides:

[0040]

Embedded image

Amplified by RT-PCR in the presence of

The resulting DNA fragment, which had been purified and digested with the restriction enzyme XbaI, was digested with pMRS-3.
0 expression vector. The resulting pMRS167 vector contains a DNA fragment containing the ATG codon, a sequence corresponding to nucleotides 1222-1497 of the EMBL sequence J05581, and two stop codons, TGA and TAA. This fragment is shown in FIG.

Example 5 Construction of Plasmid pMRS175 Plasmids pMRS166, 169, 168, 167 were replaced with the following nucleotide pairs:

[0044]

Embedded image

Were subjected to a PCR reaction in the presence of

The four DNA fragments obtained in each PCR reaction were mixed in an equimolar amount, and a PCR reaction was performed in the presence of the V11 and V10 oligonucleotides.

The resulting DNA fragment purified and digested with restriction enzyme XbaI was cloned into pMRS30 expression vector. The resulting pMRS175 vector contains a DNA fragment containing the ATG codon, a sequence corresponding to nucleotides 136-1497 of the EMBL sequence J05581, and two stop codons, TGA and TAA. This fragment is shown in FIG.

Example 6 Construction of Plasmid pMRS171 MCF7 tumor cells (ATCC HTB-22) were cultured in minimal essential Eagle's medium supplemented with 10% fetal calf serum. Trypsinize 10 million cells,
After washing with PBS, mRNA was extracted.

An aliquot of this RNA was prepared using the following synthetic oligonucleotides:

[0050]

Embedded image

Was subjected to an RT-PCR reaction in the presence of

This reaction yielded a DNA fragment called Fragment 1.

The DNA from pGEX11T (Pharmacia) was synthesized with the following synthetic oligonucleotides:

[0054]

Embedded image

The PCR reaction was performed in the presence of By this reaction, a DNA fragment called fragment 2 was obtained.

The DNA fragments 1 and 2 obtained in each PCR reaction were mixed in an equimolar amount,
PCR reactions were performed in the presence of BIup and LacDown oligonucleotides.

The resulting DNA fragment, purified and digested with restriction enzymes XbaI and BamHI, was cloned into a commercially available plasmid called pUC18. The resulting pMRS156
Vectors include a DNA fragment comprising a sequence encoding ubiquitin fused to a sequence encoding a bacterial β-galactosidase moiety. The fragment designated UBILacI is shown in FIG.

The DNA of plasmid pMRS166 was replaced with the following synthetic oligonucleotide sequence:

[0059]

Embedded image

[0060] PCR reaction in the presence of

The resulting DNA fragment, which had been purified and digested with the restriction enzymes XbaI and BamHI, was fused to the UBILacI fragment from the pMRS156 plasmid by ligation at two BamHI sites. The resulting fragment was cloned into the pMRS30 expression vector. The resulting pMRS171 vector contains UBILac
I sequence, a sequence corresponding to nucleotides 136-339 of the EMBL sequence J05581, and a DNA fragment containing the two stop codons TGA and TAA. This fragment is shown in FIG.

Example 7 Construction of Plasmid pMRS174 The DNA of plasmid pMRS169 was replaced with the following synthetic oligonucleotide sequence:

[0063]

Embedded image

A PCR reaction was performed in the presence of

The resulting DNA fragment, which had been purified and digested with the restriction enzymes XbaI and BamHI, was fused to the UBILacI fragment from the pMRS156 plasmid by ligation at two BamHI sites. The resulting fragment was cloned into the pMRS30 expression vector. The resulting pMRS174 vector is UBILac
I sequence, a sequence corresponding to nucleotides 205-720 of the EMBL sequence J05581 and a DNA fragment containing the two stop codons TGA and TAA. This fragment is shown in FIG.

Example 8 Construction of Plasmid pMRS173 The DNA of plasmid pMRS168 was replaced with the following synthetic oligonucleotide sequence:

[0067]

Embedded image

Was subjected to a PCR reaction in the presence of

The resulting DNA fragment, which had been purified and digested with the restriction enzymes XbaI and BamHI, was fused to the UBILacI fragment from the pMRS156 plasmid by ligation at two BamHI sites. The resulting fragment was cloned into the pMRS30 expression vector. The resulting pMRS173 vector is UBILac
I sequence, a sequence corresponding to nucleotides 631-1275 of EMBL sequence J05581, and a DNA fragment containing two stop codons, TGA and TAA. This fragment is shown in FIG.

The DNA of plasmid pMRS167 was replaced with the following synthetic oligonucleotide sequence:

[0071]

Embedded image

Was subjected to a PCR reaction in the presence of

The resulting DNA fragment, which had been purified and digested with the restriction enzymes XbaI and BamHI, was fused to the UBILacI fragment from the pMRS156 plasmid by ligation at two BamHI sites. The resulting fragment was cloned into the pMRS30 expression vector. The resulting pMRS172 vector is UBILac
I sequence, a sequence corresponding to nucleotides 1222-1497 of EMBL sequence J05581, and a DNA fragment containing two stop codons, TGA and TAA. This fragment is shown in FIG.

Example 10 Construction of Plasmid pMRS176 The DNA of plasmid pMRS167 was subjected to a PCR reaction in the presence of the following synthetic oligonucleotide sequences: V3 (see Example 6) and V10 (see Example 4).

The resulting DNA fragment, which had been purified and digested with the restriction enzymes XbaI and BamHI, was fused to the UBILacI fragment from the pMRS156 plasmid by ligation at two BamHI sites. The resulting fragment was cloned into the pMRS30 expression vector. The resulting pMRS176 vector contains UBILac
I sequence, a sequence corresponding to nucleotides 136 to 1497 of EMBL sequence J05581, and a DNA fragment containing two stop codons, TGA and TAA. This fragment is shown in FIG.

Example 11 Transfection and Transcription Test of Eukaryotic Cells CHO (Chinese Hamster Ovary) cells were supplemented with ribonucleotides and deoxyribonucleotides at the time of transfection.
Cultured in EM.

Dendritic cells were obtained from CD34 + hematopoietic progenitor cells cultured in IMDM supplemented with GM-CSF, IL4, CSF, Flt3 and TNFα without serum.

PBM cultured in RPMI supplemented with CSF, GM-CSF, and IL-4
Dendritic cells were obtained from monocytes isolated from C (peripheral blood mononuclear cells).

In each case, about one million cells were transfected with one of the plasmids described in Examples 1-10. Transfection was performed by lipofection using 3 μg of plasmid DNA and 4 μl of DMRIE (Gibco).

After 24 hours, cells were harvested, washed with PBS, and lysed to extract mRNA.

An aliquot of the mRNA was subjected to RT-PCR in the presence of a pair of oligonucleotides specific for the transfected DNA plasmid.

This experiment was performed using the following oligonucleotides: p11 / pMRS166 for V11 /
V12 / V6 for V4 and pMRS169, V1 for pMRS168
3 / V8, V4 / V10 for pMRS167, V4 / V10 for pMRS175, UBIup / V4 for pMRS171, UBIup / V6 for pMRS174, UBIup / V8, pM for pMRS173.
UBIup / V10 for RS172, V4 / V for pMRS176
10 was performed for each of the plasmids described in Examples 1 to 10.

As a representative example, FIG. 12 shows the DN obtained by RT-PCR from mRNA of three cell populations transfected with the pMRS169 plasmid.
3 shows the electrophoretic analysis of the A fragment. In this case, the oligonucleotide pair V12
/ V6 was used.

Example 12 Results of In Vivo Studies In the in vivo studies, a mixture of the four fragments and the pMRS30 plasmid (vector without insert, ie used as negative control) were used. To test that immunization had occurred, an ELISA test was used to show human mucin-specific antigen.

[0085] In vivo studies were performed using human MUC1 transgenic C57BL mice. As a result, in these animals, the MUC1 protein showed its own protein. The applied vaccination schedule was plasmid D
100 μg NA, 3 intradermal (back site, 50 μg DNA on each side)
) Administration (Days 0, 14, 28). On day 14 after the last dose,
Animals were sacrificed and sera were tested for anti-human mucin antibodies.

The fragment mixture assayed, ie the object of the invention, stimulated the immune response well in the treated animals.

On the other hand, a vaccination experiment was also carried out with a 60 amino acid peptide (this peptide is referred to as 3XTR), which corresponds to the three repeats of 20 amino acids from position 86 to position 105 shown in FIG.

[0088] The two types of vaccination differ in the type of antibody response elicited. Antibody titers were higher in vaccination with 3XTR. further,
The IgG subtypes noticed supported essentially humoral (antibody) responses in the case of vaccination with 3XTR, and cellular responses (cytotoxicity) in the case of vaccination with DNA. . For antitumor treatment, a cytotoxic immune response is in principle preferred. Since we experimented with transgenic mice where human mucin is "self", we can foresee a similar response in humans. This response supports the use of the compounds of the invention as DNA vaccines in the treatment of MUC1 overexpressing human tumors.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is the DNA nucleotide sequence (along with the respective amino acid sequence) inserted into the XbaI site of the pMRS166 expression vector. This DNA is preceded by the translation initiation codon ATG and followed by two translation stop codons TGA and TAA.
Includes a sequence corresponding to nucleotides 136-339 of the EMBL sequence J05581. That is, the encoded polypeptide is one of the EMBL sequences J05581.
The amino acid encoded by the 36-139 fragment contains methionine followed.

FIG. 2: Xb of pMRS30 expression vector to obtain pMRS169 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA comprises a sequence corresponding to nucleotides 205-720 of the EMBL sequence J05581, preceded by the translation initiation codon ATG and followed by two translation stop codons TGA and TAA. That is, the encoded polypeptide contains methionine followed by the amino acids encoded by the 205-720 fragment of the EMBL sequence J05581.

FIG. 3. Xb of pMRS30 expression vector to obtain pMRS168 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA contains a sequence corresponding to nucleotides 631-1275 of the EMBL sequence J05581 preceded by the translation initiation codon ATG and followed by two translation stop codons TGA and TAA. That is, the encoded polypeptide contains methionine followed by the amino acids encoded by the 631-1275 fragment of the EMBL sequence J05581.

FIG. 4. Xb of pMRS30 expression vector to obtain pMRS167 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA contains a sequence corresponding to nucleotides 1222 to 1497 of the EMBL sequence J05581 preceded by the translation initiation codon ATG and followed by two translation stop codons TGA and TAA. That is, the encoded polypeptide contains methionine followed by the amino acids encoded by the 1222-1497 fragment of the EMBL sequence J05581.

FIG. 5. Xb of pMRS30 expression vector to obtain pMRS175 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA includes a sequence corresponding to nucleotides 136 to 1497 of the EMBL sequence J05581, preceded by the translation initiation codon ATG and followed by two translation stop codons, TGA and TAA. That is, the encoded polypeptide contains methionine followed by the amino acids encoded by the 136-1497 fragment of the EMBL sequence J05581.

FIG. 6 is the DNA nucleotide sequence (along with the respective amino acid sequence) designated UBILacI. The encoded polypeptide is Chau V. et al. Science 243.
: 1576, 1989, containing a ubiquitin sequence fused to a partial sequence of a bacterial protein β-galactosidase.

FIG. 7: Xb of pMRS30 expression vector to obtain pMRS171 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA is followed by two translation stop codons, TGA and TAA.
It contains a sequence called UBILacI (see FIG. 6) fused to a sequence corresponding to nucleotides 136 to 339 of sequence J05581 of EMBL. Therefore,
The encoded polypeptide is a 136-33 of the EMBL sequence J05881.
9 comprises the amino acid sequence shown in FIG. 6 fused to a sequence containing the amino acids encoded by the nine fragments.

FIG. 8. Xb of pMRS30 expression vector to obtain pMRS174 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA is followed by two translation stop codons, TGA and TAA.
It contains a sequence called UBILacI (see FIG. 6) fused to a sequence corresponding to nucleotides 205 to 720 of sequence J05581 of EMBL. Therefore,
The encoded polypeptide is a EMBL sequence J05881 from 205 to 72.
0 comprises the amino acid sequence shown in FIG. 6 fused to a sequence containing the amino acids encoded by the 0 fragment.

FIG. 9. Xb of pMRS30 expression vector to obtain pMRS173 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA is followed by two translation stop codons, TGA and TAA.
It contains a sequence called UBILacI (see FIG. 6) fused to a sequence corresponding to nucleotides 631-1275 of sequence J05581 of EMBL. Thus, the encoded polypeptide is the EMBL sequence J05881 at 631-1.
6 includes the amino acid sequence shown in FIG. 6 fused to a sequence containing the amino acids encoded by the 275 fragment.

FIG. 10. Xb of pMRS30 expression vector to obtain pMRS173 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA is followed by two translation stop codons, TGA and TAA.
It contains a sequence called UBILacI (see FIG. 6) fused to a sequence corresponding to nucleotides 1222-1497 of sequence J05581 of EMBL. Thus, the encoded polypeptide is the 1222 of sequence J05881 of EMBL.
FIG. 6 fused to a sequence containing the amino acids encoded by the １４1497 fragment.
The amino acid sequence shown in Table 1 is included.

FIG. 11. Xb of pMRS30 expression vector to obtain pMRS176 expression vector
DNA nucleotide sequence (along with the respective amino acid sequence) inserted at the aI site. This DNA is followed by two translation stop codons, TGA and TAA.
It contains a sequence called UBILacI (see FIG. 6) fused to a sequence corresponding to nucleotides 136 to 1497 of sequence J05581 of EMBL. Thus, the encoded polypeptide is 136-1 of the EMBL sequence J05881.
6 contains the amino acid sequence shown in FIG. 6 fused to a sequence containing the amino acids encoded by the 497 fragment.

FIG. 12 is an electrophoresis analysis on a 1% agarose gel in 1 × TBE. Each pMR
Transfected in S169 and with reverse transcriptase (lanes 4, 8, 1
2) or subjected to RT-PCR reaction without reverse transcriptase (lanes 5, 9, 13)
MRNA extracted from CHO, CD34 + dendritic cells and PBMC-derived dendritic cells
It is. Molecular weight DNA marker (lane 1); in-house negative control (lanes 2, 6);
In-house positive control (lanes 3, 7, 10, 11); Promega kit positive control (lane 14).

FIG. 13 is the nucleotide sequence of the pMRS30 expression vector. The region at positions 1-2862 corresponds to the AccI (position 50) of the pSV2CAT vector (EMBL M77788).
4) corresponds to the -BamHI (position 3369) region; the 2863-3721 region contains the human cytomegalovirus promoter (human cytomegalovirus major immediate early gene enhancer); the 3722-4905 region corresponds to the XbaI (position 3727). ), And a plurality of cloning sites and a processing signal for the rabbit β-globin gene.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 26, 2001 (2001.1.26)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Desantis, Rita Italy, I-50131 Florence, Via Rizmond F-term (reference) AA01 AA20 BA31 CA04 DA03 EA04 GA11 HA17 4C084 AA02 AA03 AA06 AA13 BA01 BA02 BA21 BA22 BA23 BA44 DA01 DA27 NA14 ZB26 4C085 AA03 AA32 BB01 CC21 DD86 EE01 EE06 4C087 AA01 AA02 BB65 ZB26 NA14

Claims (20)

[Claims] 1. Combining one or more DNA molecules encoding a fragment of a protein that is overexpressed in tumor cells to induce an anti-tumor Ag-specific immune response with appropriate excipients and adjuvants A pharmaceutical composition comprising: 2. The pharmaceutical composition according to claim 1, wherein the overexpressed protein is MUC-1. 3. The pharmaceutical composition according to claim 1, comprising at least two DNA molecules each comprising a cDNA sequence encoding a mucin fragment (MUC-1). 4. The composition of claim 3, comprising at least three DNA molecules each comprising a cDNA sequence encoding a mucin fragment (MUC-1). 5. The composition of claim 4, comprising at least four DNA molecules each comprising a cDNA sequence encoding a mucin fragment (MUC-1). 6. The DNA sequence comprises from about 200 to about 700 nucleotides, each sequence being contiguous and adjacent to its neighbors at about 5 'and / or 5' ends.
The composition of any one of claims 3 to 5, wherein the composition may partially overlap from 0 to about 150 nucleotides. 7. The mixture used comprises at least two plasmid DNAs, the sequences each comprising a DNA fragment selected from those described in FIGS.
The pharmaceutical composition according to any one of claims 2 to 6, comprising a molecule. 8. The pharmaceutical composition according to claim 7, wherein the mixture used consists of a pool of plasmid DNA molecules, the sequences each comprising a DNA fragment selected from those described in FIGS. 9. The pharmaceutical composition according to claim 1, wherein a plasmid DNA molecule comprising the sequence shown in FIG. 5 is used. 10. The pharmaceutical according to any one of claims 7 to 9, wherein the plasmid DNA used is derived from a fusion between the pMRS30 expression vector of Fig. 13 and each of the sequences described in Figs. Composition. 11. The sequence used corresponding to a single fragment of said protein is 5
7. A pharmaceutical composition according to any one of claims 2 to 6, which, at the 'end, is preceded by the sequence set forth in Fig. 6, which encodes a ubiquitin and LacI moiety from Escherichia coli. 12. The mixture comprises pMRS30 as described in FIG. 13 and FIGS.
12. The pharmaceutical composition according to claim 11, consisting of one or more sequences derived from binding to a DNA sequence selected from those described in. 13. The mixture comprises pMRS30 as described in FIG. 13 and FIGS.
The pharmaceutical composition according to claim 11, consisting of the entire sequence derived from binding to a DNA sequence selected from those described in (1). 14. The pharmaceutical composition according to claim 11, wherein the mixture consists of a sequence derived from the binding between the pMRS30 expression vector and the sequence described in FIG. 15. The pharmaceutical composition according to any one of claims 1 to 14, further comprising a cytokine or a plasmid encoding the cytokine. 16. Encoding a MUC-1 protein fragment, the sequence of which is shown in FIG.
A plasmid DNA molecule consisting of a pMRS30 expression vector linked to a DNA sequence selected from the group of those described in 5 above. 17. A DNA molecule encoding a protein MUC-1 fragment, the 5 'end of which is preceded by the sequence set forth in FIG. 18. The method according to claim 17, which is selected from those described in FIGS.
The DNA fragment according to any one of the preceding claims. 19. A plasmid DNA molecule obtained by combining a pMRS30 expression vector with a DNA molecule selected from those according to claim 17 or 18. 20. The method for preparing a composition having an antitumor effect according to claim 16.
Use of a DNA molecule according to any one of claims 19 to 19.